Topoisomerase 1 (top1) is a DNA binding protein that regulates DNA topology through changing the degree of supercoiling by cutting the DNA strand. Human DNA top1 is a 100 kDa nuclear protein and is the target of an important class of anti-neoplastic agents called camptothecins (CPTs). CPTs are lethal to cells as a result of the formation of DNA-top1-CPT ternary complexes. Relatively little is known about the interaction between top1 and other proteins, even though these interactions are likely important in the cellular functions of top1 and in the cytotoxic mechanisms of camptothecins (Haluska, et al. (1998) Adv. Enz. Regul. 38:253-62). Physical interactions have been detected between top1 and two proteins implicated in carcinogenesis, SV40 T antigen and p53 (Haluska, et al. (1998) Nucleic Acid Res. 26(7) :1841-7; Zhou, et al. (1999) Gene 235(1-2):93-101). These findings highlight the potential significance of top1-binding proteins in tumorigenesis.
Using a yeast two-hybrid screen, a novel topoisomerase 1- and p53-binding protein called topors was discovered. Topors is a RING protein that binds to the N-terminus of human top1 (Haluska, et al. (1999) supra). The coding region of topors is deposited under GENBANK Accession Number AF098300. Topors is unique in that it contains both a RING finger and serine and arginine domains in the same polypeptide. Subsequent to the identification of topors, a peptide fragment of topors was identified as a p53-binding protein (Zhou, et al. (1999) supra). Homology searches indicate that the topors RING domain is similar to the RING domain of proteins involved in ubiquitin or SUMO transfer reactions.
Ubiquitination is critical to cellular function. The conjugated ubiquitin system tags proteins for degradation by proteosomes. As shown in FIG. 1, the ubiquitin activating enzyme activates ubiquitin in the presence of ATP. This enzyme is a single enzyme in most species. The ubiquitin conjugating enzyme contributes to substrate specificity. Ubiquitin ligase also confers substrate specificity and can be a complex of proteins (SCF) or a single protein (c-Cbl). Ubiquitin ligase may transfer ubiquitin directly from the ubiquitin conjugating enzyme to the substrate or form an ubiquitin-conjugate intermediate (see FIG. 1).
SUMO (small ubiquitin-related modifier) proteins are small protein tags that are conjugated to cellular regulator proteins. The regulator proteins include oncogenes and tumor suppressor genes that play key roles in the control of cell growth, differentiation and apoptosis. SUMO conjugation affects the substrates' subcellular localization and stability as well as transcriptional activities. Three different SUMO proteins are conjugated to proteins, SUMO-1, SUMO-2 and SUMO-3. SUMO-1 is conjugated to proteins as a monomer, and SUMO-2 and SUMO-3 are conjugated to proteins as higher molecular weight polymers with SUMO-1 terminating further SUMO addition.
One target of SUMO modification includes proteins involved in formation of the promyclocytic leukemia (PML) nuclear bodies. Acute promyelocytic leukemia, a type of cancer affecting the blood, is characterized by an abnormal block in the development of stem cells. Topors promotes the stability of PML nuclear bodies and perhaps alters their role in transcriptional regulation, cellular proliferation and anti-viral responses. The activity of several transcription factors is altered by sumoylation, including C/EBP proteins, c-Myb, glucocorticoid receptor, androgen receptor, and progesterone receptor. Sumoylation of topoisomerase I alters its localization in the nucleus, and histone deacetylase enzymes are targets of this system. Viral proteins are targets of sumoylation, suggesting that infection and anti-viral cellular defenses may be affected by this system. Protein sumoylation is important in cell cycle progression and genomic stability. Sumoylation may also alter the stability of proteins with polyglutamine repeats involved in neurodegenerative disorders, adding further to the important and diverse roles of this protein modification system.
Since Top1 appears to be an important potential anti-cancer drug target, it is clinically relevant to understand the function and expression of proteins, such as topors, that interact with Top1. It is further important to understand the impact of a topors antibody. The present invention relates to the characterization of topors antibody, further characterization of topors, and the methods of using both topors and topors antibody for cancer diagnostics and therapeutics.